Abstract
Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed “nuclear injection”. To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.
References
Besenfelder U, Modl J, Muller M, Brem G (1997) Endoscopic embryo collection and embryo transfer into the oviduct and the uterus of pigs. Theriogenology 47:1051–1060
Boch J et al (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512. doi:10.1126/science.1178811
Branda CS, Dymecki SM (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6:7–28
Burdon TG, Wall RJ (1992) Fate of microinjected genes in preimplantation mouse embryos. Mol Reprod Dev 33:436–442. doi:10.1002/mrd.1080330410
Burke DT, Carle GF, Olson MV (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806–812
Chan AW, Kukolj G, Skalka AM, Bremel RD (1999) Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection. Mol Reprod Dev 52:406–413. doi:10.1002/(SICI)1098-2795(199904)52:4<406:AID-MRD9>3.0.CO;2-P
Clark AJ, Bissinger P, Bullock DW, Damak S, Wallace R, Whitelaw CB, Yull F (1994) Chromosomal position effects and the modulation of transgene expression. Reprod Fertil Dev 6:589–598
Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi:10.1126/science.1231143
Couto LB, Spangler EA, Rubin EM (1989) A method for the preparative isolation and concentration of intact yeast artificial chromosomes. Nucl Acids Res 17:8010
Fujiwara Y, Miwa M, Takahashi R, Kodaira K, Hirabayashi M, Suzuki T, Ueda M (1999) High-level expressing YAC vector for transgenic animal bioreactors. Mol Reprod Dev 52:414–420
Funahashi H, Stumpf TT, Cantley TC, Kim NH, Day BN (1995) Pronuclear formation and intracellular glutathione content of in vitro-matured porcine oocytes following in vitro fertilisation and/or electrical activation. Zygote 3:273–281
Galli C, Perota A, Brunetti D, Lagutina I, Lazzari G, Lucchini F (2010) Genetic engineering including superseding microinjection: new ways to make GM pigs. Xenotransplantation 17:397–410. doi:10.1111/j.1399-3089.2010.00590.x
Garrick D, Fiering S, Martin DI, Whitelaw E (1998) Repeat-induced gene silencing in mammals. Nat Genet 18:56–59. doi:10.1038/ng0198-56
Giraldo P, Montoliu L (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res 10:83–103
Gun G, Kues WA (2014) Current progress of genetically engineered pig models for biomedical research. BioRes Open Access 3:255–264. doi:10.1089/biores.2014.0039
Karpen GH (1994) Position-effect variegation and the new biology of heterochromatin. Curr Opin Genet Dev 4:281–291
Kato M, Yamanouchi K, Ikawa M, Okabe M, Naito K, Tojo H (1999) Efficient selection of transgenic mouse embryos using EGFP as a marker gene. Mol Reprod Dev 54:43–48. doi:10.1002/(SICI)1098-2795(199909)54:1<43:AID-MRD6>3.0.CO;2-N
Kikuchi K et al (1998) Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C. Theriogenology 50:615–623
Kim BK, Cheon SH, Lee YJ, Choi SH, Cui XS, Kim NH (2003) Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa. Zygote 11:261–270
Klymiuk N et al (2012a) First inducible transgene expression in porcine large animal models. FASEB J 26:1086–1099. doi:10.1096/fj.11-185041
Klymiuk N et al (2012b) Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis. J Mol Med (Berl) 90:597–608. doi:10.1007/s00109-011-0839-y
Klymiuk N et al (2012c) Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice. Diabetes 61:1527–1532. doi:10.2337/db11-1325
Klymiuk N et al (2013) Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle. Hum Mol Genet 22:4368–4382. doi:10.1093/hmg/ddt287
Knust B, Bruggemann U, Doerfler W (1989) Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation. J Virol 63:3519–3524
Kurome M et al (2003) Comparison of electro-fusion and intracytoplasmic nuclear injection methods in pig cloning. Cloning Stem Cells 5:367–378. doi:10.1089/153623003772032862
Kurome M et al (2013) Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set. BMC Biotechnol 13:43. doi:10.1186/1472-6750-13-43
Kurome M, Kessler B, Wuensch A, Nagashima H, Wolf E (2015) Nuclear transfer and transgenesis in the pig. Methods Mol Biol 1222:37–59. doi:10.1007/978-1-4939-1594-1_4
Luo Y, Lin L, Bolund L, Jensen TG, Sorensen CB (2012) Genetically modified pigs for biomedical research. J Inherit Metab Dis 35:695–713. doi:10.1007/s10545-012-9475-0
Miyagawa S et al (2015) Generation of alpha1, 3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs. J Reprod Dev 61:449–457. doi:10.1262/jrd.2015-058
Niemann H, Kues W, Carnwath JW (2005) Transgenic farm animals: present and future. Rev Scie Tech 24:285–298
Osborn MJ et al (2013) High-affinity IgG antibodies develop naturally in Ig-knockout rats carrying germline human IgH/Igkappa/Iglambda loci bearing the rat CH region. J Immunol 190:1481–1490. doi:10.4049/jimmunol.1203041
Ostrup O et al (2009) Nuclear and nucleolar reprogramming during the first cell cycle in bovine nuclear transfer embryos. Cloning Stem Cells 11:367–375. doi:10.1089/clo.2008.0076
Palmer TD, Rosman GJ, Osborne WR, Miller AD (1991) Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci USA 88:1330–1334
Petersen B, Niemann H (2015) Molecular scissors and their application in genetically modified farm animals. Transgenic Res 24:381–396. doi:10.1007/s11248-015-9862-z
Porteus MH, Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300:763. doi:10.1126/science.1078395
Prather RS, Lorson M, Ross JW, Whyte JJ, Walters E (2013) Genetically engineered pig models for human diseases. Annu Rev Anim Biosci 1:203–219. doi:10.1146/annurev-animal-031412-103715
Renner S et al (2013) Permanent neonatal diabetes in INS(C94Y) transgenic pigs. Diabetes 62:1505–1511. doi:10.2337/db12-1065
Richter A et al (2012) Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig. BMC Biotechnol 12:84. doi:10.1186/1472-6750-12-84
Robl JM, Wang Z, Kasinathan P, Kuroiwa Y (2007) Transgenic animal production and animal biotechnology. Theriogenology 67:127–133. doi:10.1016/j.theriogenology.2006.09.034
Rogers CS (2016) Genetically engineered livestock for biomedical models. Transgenic Res. doi:10.1007/s11248-016-9928-6
Sabl JF, Henikoff S (1996) Copy number and orientation determine the susceptibility of a gene to silencing by nearby heterochromatin in Drosophila. Genetics 142:447–458
Schedl A, Montoliu L, Kelsey G, Schutz G (1993) A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature 362:258–261. doi:10.1038/362258a0
Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA 89:8794–8797
Somfai T et al (2009) Live piglets derived from in vitro-produced zygotes vitrified at the pronuclear stage. Biol Reprod 80:42–49. doi:10.1095/biolreprod.108.070235
Suzuki S et al (2012) Il2rg gene-targeted severe combined immunodeficiency pigs. Cell Stem Cell 10:753–758. doi:10.1016/j.stem.2012.04.021
Swindle CS, Klug CA (2002) Mechanisms that regulate silencing of gene expression from retroviral vectors. J Hematother Stem Cell Res 11:449–456. doi:10.1089/15258160260090915
Takahashi R, Ueda M (2010) Generation of transgenic rats using YAC and BAC DNA constructs. Methods Mol Biol 597:93–108. doi:10.1007/978-1-60327-389-3_7
Takahashi R, Ito K, Fujiwara Y, Kodaira K, Kodaira K, Hirabayashi M, Ueda M (2000) Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA. Exp Anim 49:229–233
Umeyama K et al (2013) Production of diabetic offspring using cryopreserved epididymal sperm by in vitro fertilization and intrafallopian insemination techniques in transgenic pigs. J Reprod Dev 59:599–603
Van Keuren ML, Gavrilina GB, Filipiak WE, Zeidler MG, Saunders TL (2009) Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Res 18:769–785. doi:10.1007/s11248-009-9271-2
Verma IM, Somia N (1997) Gene therapy—promises, problems and prospects. Nature 389:239–242. doi:10.1038/38410
Wall RJ, Pursel VG, Hammer RE, Brinster RL (1985) Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei. Biol Reprod 32:645–651
Wang D et al (2014) Genomic imprinting analysis of Igf2/H19 in porcine cloned fetuses using parthenogenetic somatic cells as nuclear donors. Biotechnol Lett 36:1945–1952. doi:10.1007/s10529-014-1572-8
Watanabe M et al (2012) The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer. Transgenic Res 21:605–618. doi:10.1007/s11248-011-9561-3
Watanabe M et al (2015) Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum. J Reprod Dev 61:169–177. doi:10.1262/jrd.2014-153
Whitworth KM, Prather RS (2010) Somatic cell nuclear transfer efficiency: how can it be improved through nuclear remodeling and reprogramming? Mol Reprod Dev 77:1001–1015. doi:10.1002/mrd.21242
Whyte JJ, Prather RS (2011) Genetic modifications of pigs for medicine and agriculture. Mol Reprod Dev 78:879–891. doi:10.1002/mrd.21333
Wuensch A et al (2014) Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs. Transplantation 97:138–147. doi:10.1097/TP.0b013e3182a95cbc
Xing X, Magnani L, Lee K, Wang C, Cabot RA, Machaty Z (2009) Gene expression and development of early pig embryos produced by serial nuclear transfer. Mol Reprod Dev 76:555–563. doi:10.1002/mrd.20974
Yang P et al (2008) Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin. PLoS ONE 3:e3453. doi:10.1371/journal.pone.0003453
Acknowledgements
We would like to thank Dr. Hiroshi Nagashima, Dr. Gorge Arnord, Tuna Guengoer, Ingrid Kola and Tatiana Schroeter for their invaluable technical support. This work was financially supported by the German Research Council (TRR 127 ‘Biology of xenogeneic cell, tissue and organ transplantation—from bench to bedside’) and by EXIST Forschungstransfer 03FABY064. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors are members of COST Action BM1308 “Sharing Advances on Large Animal Models—SALAAM”.
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MK, SL, BF and EW conceived and designed the study. MK, SL, BK, EK, EJ, NK, VZ performed the experiments and MK and BK drafted the manuscript. All authors read and approved the final manuscript.
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Kurome, M., Leuchs, S., Kessler, B. et al. Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs. Transgenic Res 26, 309–318 (2017). https://doi.org/10.1007/s11248-016-0004-z
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DOI: https://doi.org/10.1007/s11248-016-0004-z